N-demethylation accompanies α-hydroxylation in the metabolic activation of tamoxifen in rat liver cells

Previous work has shown that a major route of activation of tamoxifen to DNA-binding products in rat liver cells is via a-hydroxylation leading to modification of the N-2-position of guanine in DNA and to a lesser extent the N-6-position of adenine, Improved resolution by HPLC has now identified two major adducts in rat liver DNA, one of them the aforementioned tamoxifen-N-2-guanine adduct and the other the equivalent adduct in which the tamoxifen moiety has lost a methyl group. Treatment of rats or rat hepatocytes with N-desmethyltamoxifen gave rise to the second adduct, whereas treatment with tamoxifen or alpha-hydroxytamoxifen gave rise to both, Furthermore, N,N-didesmethyltamoxifen was found to be responsible for an additional minor DNA adduct formed by tamoxifen, a-hydroxytamoxifen and N-desmethyltamoxifen. The involvement of metabolism at the a position was confirmed in experiments in which [alpha-D-2-ethyl]tamoxifen, but not [beta-D-3-ethyl]tamoxifen, produced reduced levels of DNA adducts, Tamoxifen N-oxide and alpha-hydroxytamoxifen N-oxide also gave rise to DNA adducts in rat liver cells, but the adduct patterns were very similar to those formed by tamoxifen and alpha-hydroxytamoxifen, indicating that the N-oxygen is lost prior to DNA binding. These and earlier results demonstrate that in rat liver cells in vivo and in vitro, Phase I metabolic activation of tamoxifen involves both alpha-hydroxylation and N-demethylation, which is followed by Phase II activation at the alpha-position to form a highly reactive sulphate. Detection of tamoxifen-related DNA adducts by P-32-postlabelling is achieved with >90% labelling efficiency.